HPLC-based device and method for separating high complex substance mixtures

ABSTRACT

The invention concerns an HPLC-based device and method for separating high complex substance mixtures. Plant and microbial extracts are high complex substance mixtures. They contain large amounts of extremely polar and non-polar materials. In principle, said mixtures can be separated by using a chromatic method. However, separation with existing chromatographic devices, for instance HPLC installations, is extremely time-consuming. The invention seeks to create a HPLC installation that separates fully automatically high complex substances in a very short time in such a way that said substances are broken down into their components in an almost pure state and can then be fed into a test system. To this end, said HPLC-based device comprises separation column units (A, F),fractionating column units (E, G), detector units (7, and 20, 21), pumping units (B, C, D) and fraction collecting units. These units, including all separating or fractionating columns, are interconnected and controlled by multiple way valves and by a computer unit that ensures the software-controlled operational interaction of the device.

REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/EP97/05093 filed Sep. 17, 1997.

The invention relates to an HPLC-based apparatus and method forseparating highly complex mixtures of substances.

BACKGROUND OF THE INVENTION

More than one third of the medicinal drugs currently on the marketcontain active ingredients, which nature has made available, that is,they were isolated from plants or microorganisms or, at the very least,modified on this basis.

In spite of this relatively large number of biological active substance,which nature has made available, worldwide efforts have, until now, beenconcentrated more on the chemical synthesis than on the so-called poolof natural products. In recent years, however, new active ingredientswere discovered, which were created by nature and, as a result, naturalproducts chemistry or natural products biochtechnology has thereforeexperienced a renaissance.

At the same time, with the discovery or production of new activeingredients, there was a rapid development in the sector of test systemcapacities. Whereas such biological assays for finding potentially newactive ingredients some years ago required a few 100 mg of substanceand, with that, only a few throughputs of tests per year were possible,the situation at the present time is basically different. As a result oftest designs, which assume, for example, the inhibition of a specificenzyme as a measure of the biological activity, miniaturized automatictest machine can be realized, with which a million substances per yearcan be investigated, while at the same time little substance isconsumed. The presence of these enormous test system capacities meetsthe requirements of natural products research, because frequently, untilit is possible to detect a special biological activity, only a fewmilligrams of pure natural products, isolated from plants or microbialfermentations, are available.

Although a large number of natural products is already known, it must beassumed that nature has on hand a much larger number of substances,which until now have not been known, so that a high throughput screeningof a large number of crude plant and microbial extracts cannot beavoided.

However, the testing of natural products requires a protracted procedureof preliminary purification, preliminary separation, intermediatepurification and final purification, which must always be interruptedonce again by testing for biological activity. This procedure requiresmuch time, a large effort by personnel and a large logistic expenditureand moreover frequently leads to chemical substances, which are notworthwhile following up further.

In view of the cost pressures, which are carried over from the PublicHealth Service into the researching facilities, such time losses lead toconstantly greater disadvantages for research and development, based onnatural products research. Plant and microbial extracts are highlycomplex mixtures of substances. They contain extremely polar as well asnonpolar materials in large numbers. Basically, the separation of thesemixture is possible with chromatographic methods. However, the timerequired for the separation with previously known chromatographicequipment, such as HPLC equipment, is unjustifiably high.

In the BEO 1994 annual report of the Bundesministerium fur Bildung,Wissenschaft, Forschung und Technology (Federal Ministry for Education,Science, Research and Technology) pages 413 and 414, HPLC equipment forthe isolation of natural products is described, which is intended tofractionate plant and microbial extracts coarsely and finely.

The equipment has the following disadvantages:

The fraction-collecting columns, named were, are connected over 12-wayvalves, the use of which for preparative applications is very expensive.The frequency of the switching required here leads to more rapid wear ofcomponents and seals.

A variable use, corresponding to the mixture that is to be separated, byincreasing or decreasing the number of columns, is not possible, thatis, because of this design, a modular construction of the equipment isnot possible.

The large number of fraction-collecting columns leads to a long runningtime and to a high consumption of solvents.

An inexpensive and time-saving roll-over operation is not possible.

The isocratic separation in the second separation step, which isprovided for here, also leads to a disadvantageous extension of therunning time.

SUMMARY OF THE INVENTION

It is now an object of the invention to offer HPLC equipment, whichseparates highly complex mixtures of substances fully automatically in avery short time to such an extent, that the components are present in analmost pure form and can then be supplied to the test system.

This objective is accomplished with an apparatus and a method, which arebased on HPLC and defined in claims 1 and 22.

The technology of separating the extracts, which forms the basis of theinvention, is high-pressure liquid chromatography, which is in aposition to separate relatively polar as well as nonpolar compounds.Because of the large number of substances in a complex mixture ofsubstances, such as in plant and microbial extracts, the separationcannot be carried out in one step. Rather, the inventive combination ofseveral systems of separating columns is necessary, in order to achievea separation in a justifiable time.

The invention has various advantages. For example, the invention enablescoarse and fine separation to be carried out in one piece of equipmentand separated fractions to be stored on an interim basis on solid phasesfrom which they can be recovered at any time, so that practically acomplete separation of all substance fractions can be achieved within avery short time by means of software-controlled equipment. By thesemeans, it is conceivable that, if the infrastructure is suitable, thatis, if appropriate test systems, associated with a structureclarification system, are present, an identification of the effectivecomponents of an extract within 2 to 3 days becomes possible. This meansan extreme acceleration of the process of finding active ingredients, aprocess which, starting out from mixtures of natural products such asplant or microbial extracts, usually takes months. Advantageously, theinventive apparatus has a modular construction, which enables expansionsto be made, depending on the complexity of the mixtures of substances,which are to be separated.

BRIEF DESCRIPTION OF THE DRAWING

The invention is described in greater detail by means of a drawing, inwhich

FIG. 1 shows the construction and flow diagram of the apparatus.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The multi-component mixture, which is to be separated (such as a plantor microbial extract, etc.) is dissolved in methanol and mixed with RP-4material (particle size about 40 μm) in the ratio by weight of 1 part ofextract to 3 parts of RP-4 material. The solvent is removed from thismixture in a rotary evaporator, so that a flowable mixture of extractand RP-4 material is formed. The mixture is filled dry into feedingcolumn 1, which is installed in unit A, which holds the separatingcolumns.

The air is removed from the feeding column 1, which was filled dry, withthe help of a pump 2 and water as eluant over the 3-way valves 16, 17and over a 6-way valve 3. When the air has been removed, the separatingprogram, which is controlled by software, is started.

A gradient from 0% to 100% of the solvent or of the elements, conveyedwith pump 5, is run 60 minutes with a pump 4 and a pump 5 of the pumpunit B. Pump 4 pumps an aqueous buffer solution and pump 5 pumps amethanol solution. The components of the extract are flushed as afunction of their polarity from the feeding column 1 over the 6-wayvalve 3 onto the separating column 6. The separating column 6 is filledwith RP-4 material. The components are detected in a UV detector 7 andrecorded with the software. The components reach a T piece 8 where wateris metered into the eluant over pump 2 and the 3-way valves 16, 17 andthe polarity of the solution is increased by these means. After that,this eluate passes through a 6-way valve 9 and reaches a fractionatingcolumns unit E, which consists of 18 fractionating columns.

The fractionating columns of the fractionating columns unit E are filledwith different adsorbents, at which the components are extracted bysolid phase extraction.

Each fractionating column is connected for a period of 3 to 4 minutes.The fractionating columns are connected into the eluant stream overrespective 4-way valves 18.1 to 18.18. By these means, the 60-minutegradient is divided into 18 fractions. The component-free eluate reachesthe waste through the 6-way valve 9.

Each of the individual fractions, stored on the 18 fractionatingcolumns, is separated further over one of the 6 separating columns of aseparating columns unit F. At the same time, the components are backflushed from one of the fractionating columns by pump 4 and pump 5 ofthe pump unit B, over the 3-way valve 13, the 6-way valve 9 and over thecorresponding 4-way valve 18.1 to 18.18 onto one of the 6 separatingcolumns of the separating columns unit F and the components areseparated further. The 6 separating columns are connected overappropriate 4-way valves 19.1 to 19.6.

After the separating column F, the separated components reach a splittervalve 15, which supplies a portion of the volume flowing (approximately1/40) to a light-scattering detector 20. The remaining volume flowingpasses through a UV detector and reaches a T piece 22, where water isadded by the pump 2, and through a 3-way valve 16 to the eluate and thepolarity of the solution is increased by these means. This eluate thenreaches a fractionating column unit G, which is connected over ten 4-wayvalves 14.1 to 14.10 and coated with the separated components, thecomponents being extracted from the eluate by the column material. Thesevalves are controlled by a combination of identifying peaks by thedetectors 20, 21 and by time control.

The valves are controlled by the control program in such a manner, that,when the first fractionating column is charged, methanol is pumped withthe help of a pump 26 of a pumping unit D over a 3-way valve 27 throughthe corresponding 4-way valve 25.1 onto the first fractionating columnand the components are flushed through the 3-way valves 28, 29, 30 intoone of the fraction collectors 31, 32, 33 of the fraction collectingunit H. The fractionating column, rinsed clean, is conditioned withwater over the 3-way valve 27 by means of the pump 26 and over theappropriate 4-way valve 28.1 for the next fractionation.

By these means, more than 10 fractions can be processed, because thefractionation is carried out simultaneously on the fractionating columnsand the fractionating columns are also flushed and conditioned and thusprepared for a further fractionation.

    ______________________________________                                        List of Reference Symbols                                                     ______________________________________                                        1.          feeding column                                                    2.          pump                                                              3.          6-way valve                                                       4.          pump (B)                                                          5.          pump (B)                                                          6.          separating column                                                 7.          UV detector                                                       8.          T piece                                                           9.          6-way valve                                                       13.         3-way valve                                                       15.         splitter valve                                                    16.         3-way valve                                                       17.         3-way valve                                                       18.         4-way valve (18.1-18.18)                                          19.         4-way valve (19.1-9.6)                                            20.         light scattering detector                                         21.         UV detector                                                       22.         T piece                                                           24.         4-way valve (24.1-24.10)                                          25.         4-way valve (25.1-25.10)                                          26.         pump (D)                                                          27.         3-way valve                                                       28.         3-way valve                                                       29.         3-way valve                                                       30.         3-way valve                                                       31.         fraction collector                                                32.         fraction collector                                                33.         fraction collector                                                A)          separating column unit                                            B)          pump unit                                                         C)          pump unit                                                         D)          pump unit                                                         E)          fractionating columns unit                                        F)          separating columns unit                                           G)          fractionating columns unit                                        H)          fractionating columns unit                                        ______________________________________                                    

What is claimed is:
 1. An apparatus, based on HPLC, for separatinghighly complex mixtures of substances, comprisingseparating columnsunits (A, F) fractionating columns units (E, G) detector units (7, 20,21) pump units (B, C) fraction collector units (H) and a computer unitfor the software-controlled functional collaboration of the apparatus,wherein the fractionating columns of the fractionating columns unit (E)and the separating columns of the separating columns unit (F) each havea 4-way valve (18.1-18.18 and 19.1-19.6), that the fractionating columnsof the fractionating columns unit (G) each have two 4-way valves(24.1-24.10 and 25.1-25.10) and the 4-way valves (24.1-24.10,25.1-25.10) of the fractionating columns unit (G) are connectedselectably with a further pumping unit (D), wherein the separatingcolumn units (A, F) and fractionating column units (E, G) are disposedalternately.
 2. The apparatus of claim 1, wherein it has at least twoseparating column units (A, F).
 3. The apparatus of claim 1, wherein ithas at least two fractionation units (E, G).
 4. The apparatus of claim1, wherein it has at least one detector unit (20, 21).
 5. The apparatusof claim 1, wherein it has at least three pump units (B, C, D).
 6. Theapparatus of claim 1, wherein a separating columns unit (A) contains afeeding column and a separating column, connected in series, and thefurther separating column units (F) comprise at least two separatingcolumns.
 7. The apparatus of claim 1, wherein the separating columnsunit (A) consists of a feeding loop and a separating column.
 8. Theapparatus of claim 1, wherein at least two detector units (7, 20, 21)are contained, which are disposed between separating column units (A, F)and fractionating units (E, G).
 9. The apparatus of claim 1, wherein ithas at least one detector unit (20, 21) comprising a detector that isselective and a detector that is not selective.
 10. The apparatus ofclaim 1, wherein the detectors are UV and light-scattering detectors.11. The apparatus of claim 1, wherein it has a mass-selective detector,such as a mass spectrometer.
 12. The apparatus of claim 1, wherein atleast three pumping units (B, C, D) are contained, at least one pumpingunit (B) having at least two high pressure gradient pumps (4-5).
 13. Theapparatus of claim 1, wherein the pumping units (B, C, D) are connectedover multi-way valves with the fractionating column units (E, G) and theseparating columns units (A, F).
 14. The apparatus of claim 1, whereinthe separating columns of the separating column units (A, F) are filledwith reverse phase materials (RP).
 15. The apparatus of claim 1, whereinthe separating columns of the separating columns unit (A, F) and thefractionating columns of the fractionating columns unit (E, G) arefilled with normal and reverse phase materials.
 16. The apparatus ofclaim 1, wherein the separating columns and fractionating columns arefilled with silica gel, with modified silica gels and/or with polymerphases.
 17. The apparatus of claim 1, wherein the pumping unit (B), theseparating columns unit (A) and the fractionating columns unit (E) areselectably connected with one another over a 6-way valve (3).
 18. Theapparatus of claim 1, wherein the fractionating columns unit (E) and theseparating columns unit (F) are connected controllably with one anotherover a 6-way valve (9).
 19. An HPLC-based method for separating highlycomplex mixtures of substances, comprising the following steps:gradientseparation of a highly complex mixture of substances in a firstseparating columns unit (A) into a defined number of fractions by meansof a pump unit (B) increasing the polarity of the eluant by the additionof water by means of a pumping unit (C) transferring the fractions to afirst fractionating columns unit (G), the number of columns of whichcorresponds to the number of separated fractions, and absorbing thepreviously separating fractions on a fractionating column each by solidphase extraction sequentially flushing the fractions, absorbed in thefirst fractionating columns unit (E), with less polar eluants into asecond separating columns unit (F) increasing the polarity of the eluantby adding water over a pumping unit (C) sequentially transferring thefractions, which have been separated further, in accordance with thesignals of the detector unit (20, 21) into a fractionating columns unit(G) and absorbing each fraction on a fractionating column by solid phaseextraction, wherein there is a further fractionation of the secondseparating columns unit (F) with the weak gradient by means of the pumpunit (B), that there is a sequential flushing of the fractions from thefractionating columns unit (G) into the fraction collector unit (H) bymeans of a pump unit (D) and a less polar eluant and a subsequentconditioning of the fractionating column, flushed clean, by means of thepump unit (D), and that there is transport of the mobile phase and thereare conditioning steps or equilibrating steps of the individual columnsin the separating columns unit, which are required in the interim, bycontrolling the pump units (B, C, D), by switching the multi-way valvesand controlling the fraction collector while processing the signals ofthe detector units (7 and 20, 21) using a computer unit.
 20. The methodof claim 19, wherein the addition of the mobile phase to each individualseparating column of the separating columns unit (F) and each individualfractionating column of the fractionating columns unit (E, G) is carriedout separately, under the control of a computer, over 4-way valves. 21.The method of claim 19, wherein, after a fractionating column of thefractionating columns unit (G) in a fraction collector (31, 32, 33) ofthe fraction collector unit (H) is flushed to clean it by means of thepump unit (D), the fractionating column is conditioned for the nextfractionation.
 22. The method of claim 19, wherein the highly complexmixture of substances is supplied to the apparatus over a supplyingcolumn.
 23. The method of claim 19, wherein the highly complex mixtureof substances is mixed with an adsorbent and suspended in a solvent suchas methanol, after which the solvent is removed and the adsorbent,charged with the complex mixture of substances, is filled into thesupplying column.
 24. The method of claim 19, wherein the separation ofthe mixture of substances in the separating columns unit (A) isaccomplished with a gradient of increasing lipophilicity.
 25. The methodof claim 19, wherein aqueous buffer solutions and lipophilic solventsare used as eluants.
 26. The method of claim 19, wherein solvents fromthe group consisting of, are used as lipophilic solvents.
 27. The methodof claim 19, wherein a peak identification program is used, whichpermits the number of fractions to be optimized.
 28. The method of claim19, wherein different adsorbents, corresponding to the polarity of thefraction, are used in the columns of the fractionating columns unit (E,G).
 29. The method of claim 19, wherein the fractionating columns arecleaned by back flushing.